Methods for detection of rearranged DNA

ABSTRACT

The invention provides methods for the identification of members of a malignant lymphocyte clone by analysis of clonotypic DNA rearrangements of T cell or B cell receptor genes. The DNA or RNA from isolated single tumor cells is amplified by PCR using consensus primers to the VDJ region of the receptor genes, and the sequence of the VDJ region is obtained from each. The clonotypic sequence of the malignant clone is identified as the most frequent VDJ sequence amplified. Individual-specific PCR primers for the VDJ region are then designed based upon the clonotypic sequence. These specific PCR primers are used to detect and quantitate clonotypic cells in subsequent patient samples using in situ PCR or in situ RT-PCR. Fractionated or unfractionated samples of blood or bone marrow, as well as tissue sections can be analyzed. The methods provide a highly sensitive and quantitative means to monitor the progress of disease and the efficacy of treatment protocols, as well as to detect members of the malignant clone in autologous bone marrow cells destined for transplant.

RELATED APPLICATION

This application is the National Stage of International application No.PCT/US97/09534, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Serial No. 60/019,106, filed on Jun. 3, 1996,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of Invention

The invention relates generally to a method for the detection ofmalignant cells, and the use of the method to monitor diseaseprogression and response to treatment in cancer patients. In particular,the invention relates to the identification of malignant lymphocytes byPCR amplification of immunoglobulin or T cell receptor genes which areuniquely rearranged in the malignant clone.

2. Description of the Related Art

Lymphoid malignancies are characterized by the proliferation of cellswhich carry a unique genetic marker by virtue of their rearrangedreceptor genes, the immunoglobulin (Ig) genes in B cells, and the T cellreceptor (TCR) genes in T cells. It is well known in the art that germline genes for both Ig and TCR exist as pools of gene segments whichbecome assembled during the normal differentiation of B and Tlymphocytes by a process of site specific recombination (Alberts et.al., 1995). Diversity of Ig and TCR are generated by the combinatorialassociation of these gene segments from the different pools, so that thetotal repertoire of antigen receptors is log-folds greater than theactual number of receptor gene segments.

The Ig genes comprise 3 clusters of genes, on three differentchromosomes: (1) the heavy chain (IgH) genes, which include theimmunoglobulin heavy chains, (2) k light chain genes and (3) l lightchain genes, which encode the immunoglobulin light chains. The IgHcluster consists of four pools of gene segments, known as C (constant),J (joining), D (diversity) and V (variable). There are nine C genesegments, arranged in an ordered cluster, which determine the class ofthe heavy chain. The variable region of the IgH is encoded by poolscomprising 6 J segments, 10 or more D segments and at least 50 Vsegments. The arrangement of these gene segments on the chromosome isdepicted in

FIG. 1. The Ig light chain genes consist of C, J, and V, but no Dsegment. Similarly, the genes for the a and b and the g and d chains ofthe TCR exist on separate chromosomes as pools of C, J, D, and V genesegments.

During differentiation of a T or B cell, the germ line genes arerearranged so that one member of each pool of variable region genesegments (J, D, and V) is randomly selected and the selected segmentsare joined together. The process of site specific recombination thattakes place during lymphocyte differentiation is distinctive, in thatduring the joining of the Ig and TCR gene segments J, D and V, avariable number of nucleotides may be lost from the ends of therecombining gene segments. In the case of IgH recombination only, one ormore nucleotides may also be randomly inserted at the joining site. Thisloss and gain of nucleotides at the joining sites Ig or TCR is a sourceof further diversity. It yields rearranged Ig or TCR genes which may bedifferent in length. If short regions spanning the junctions of genesegments (VD, DJ, and JC) are examined, they may be substantiallydifferent in length.

The variable regions of both Ig and TCR comprise three regions withlittle sequence homology between different clones, which are known as“hypervariable” or “complementarity-determining regions” (known as CDR1,CDR2, and CDR3, shown in FIG. 1). The intervening portions of thevariable region are more consistent between different clones, and areknown as “framework” (FR1, FR2, FR3, and FR4). The CDR3 region, which isencoded by the VJ junctional region of the light chain, and the D regionplus the VD and DJ junctional regions of the heavy chain, is the mosthighly variable, due to the somatic mutations introduced duringrecombination, as described above.

Each B lymphocyte expresses only a single rearranged IgH gene, and asingle rearranged k or l gene. Each mature T lymphocyte expresses asingle TCR a chain and a single TCR b chain. In a lymphoid malignancy,if clonal expansion of a tumor progenitor cell took place afterrearrangement of the Ig or TCR gene, it is possible to identify asignature or clonotypic rearrangement which is characteristic of themalignant clone. With the appropriate molecular probes, cells related tothe malignant clone can be distinguished from cells with unrearranged Igor TCR genes, or cells which carry different rearrangements. Therearrangement of Ig or TCR genes in a clone is called its “clonotypicrearrangement”.

It is clinically important to be able to detect and characterize tumorcells, not only at diagnosis, but during and after treatment. It is alsoimportant to be able to detect any malignant cells that mightcontaminate a population of stem cells destined for autologoustransplantation after ablative chemotherapy. The following methods whichare currently available to detect malignant cells carrying a monoclonalor clonotypic rearrangement in patient samples differ in their accuracyand their sensitivity. None are quantitative.

Morphological examination of cells in patient blood samples or biopsiesis currently used, but is relatively insensitive in detecting minimalresidual disease. Also, cells which might be related to the malignantclone, but are at a different stage of maturation, and thus have adifferent morphology from the bulk of the tumor cells, are probablymissed.

Southern blot hybridization analysis of isolated DNA, a technique whichis well known in the art, requires that between 1 and 5 percent of thecells in the patient sample carry a clonotypic rearrangement for it tobe detected. Although this technique has been used to detect amonoclonal population of cells which are present in high frequency, itis not useful for the detection of minimal residual disease, because itis not sensitive enough. Also, it cannot provide sequence informationthat definitively characterizes a malignant clone.

Recently, techniques have been developed which rely upon the use of thepolymerase chain reaction (PCR) to amplify clonotypic DNA rearrangementsin malignant cells. PCR, which is well known in the art (U.S. Pat. No.4,683,202 to Mullis, 1987), is a process of repeated cycles of DNAdenaturation, followed by DNA synthesis which is used to amplifysegments of DNA between two fixed anchor points on a DNA molecule.

Single stranded oligonucleotide primers, called PCR primers, areconstructed (based on previously obtained nucleic acid sequenceinformation) which will hybridize to the anchor points, one primer, theupstream primer, on the sense strand, and the other primer, thedownstream primer, on the antisense strand. The DNA segment is heatdenatured, and then cooled to a temperature at which the PCR primerswill anneal to their complementary sequences on the DNA segment. Aheat-stable DNA polymerase enzyme then copies the DNA between the twoanchor points. In 20-40 or more successive cycles of denaturation andDNA synthesis, the DNA segment of interest (between the two anchorpoints) can be amplified a million-fold or more. The two anchor pointsmust be within a few thousand nucleotides of each other for efficientamplification to occur. In RT-PCR (reverse transcriptase PCR), the RNAin cells is used as the template. It is first copied into cDNA using theenzyme reverse transcriptase, and the resultant cDNA is subjected to thePCR reaction.

In the context of clonotypic rearrangements, “consensus” or “framework”PCR primers which hybridize to DNA in the constant or framework regionsof rearranged Ig or TCR are used to amplify DNA prepared from patientblood or bone marrow samples containing a high proportion of tumorcells. For example, the upstream primer might be chosen from the 5′ endof the V segment, and the downstream primer from the J segment. Germline (unrearranged) DNA will not be amplified to detectable levelsbecause the distance between the primers is too great for efficientsynthesis. A monoclonal rearrangement can often (but not always) bedetected as a single band if the amplified DNA is electrophoresed on anappropriate gel. This amplified DNA represents the putativehypervariable region containing the clonotypic V(D)J rearrangement. Morespecific PCR primers, referred to as patient-specific PCR primers can bedesigned once the clonotypic sequence has been determined. The followingprior art utilizes PCR technology.

U.S. Pat. No. 5,418,132 to Morley (1995) teaches a method for thediagnosis of leukemia and lymphoma by PCR amplification of Ig or TCRgene segments using consensus framework primers, followed by separationof the PCR products on the basis of size. Because rearranged Ig or TCRgenes vary somewhat in their size, as noted above, a clonotypicrearrangement that has been amplified can often be detectable as adiscrete band on a gel. If the patient sample does not contain amonoclonal population of cells, size separation will yield a smear, withno detectable discrete band. However, the inventors disclose that thismethod fails to produce a discrete band with every patient sample, evenwhen multiple pairs of primers are employed. This method could be usefulduring initial diagnosis when the tumor burden is high, but is notproposed as a means of following minimal residual disease aftertreatment, because it does not involve detection of a specificclonotypic rearrangement.

Flow cytometry-based fluorescent in situ hybridization (FISH) usingimmunoglobulin heavy chain variable region probes (Cao et al., 1995a;1995b) has been suggested as a method to detect clonotypicrearrangements in individual cells in myeloma. The FISH techniqueinvolves hybridization of a biotin-labeled anti-sense RNA probe to theunamplified RNA in fixed cells in suspension. Cells which contain RNAcomplementary to the clonotypic sequence are then detected by means offlow cytometry. The sequence of the anti-sense RNA probes are derived byamplifying the mRNA expressed in myeloma patients' bone marrowmononuclear cells using RT-PCR from homogenized total RNA with consensusframework PCR primers for the IgH variable region. Because the RNA inthe cells to be assayed is not amplified, this method is only sensitiveenough to detect cells in which the clonotypic RNA is highly abundant,but it cannot detect cells with a low level of the RNA, or cells inwhich the DNA is rearranged, but is not being transcribed. For example,FISH might work well with myeloma plasma cells, which are virtual“immunoglobulin factories”, and contain extremely high concentrations ofmRNA encoding the Ig being produced by the cell. However, FISH would notbe sensitive enough to detect pre-B cells or B cells which share theclonotypic rearrangement of a myeloma malignant clone, but do nottranscribe or have a low level transcription of the gene.

RT-PCR or PCR using patient-specific PCR probes have been used toamplify bulk RNA or DNA preparations made from myeloma patient samplesin an attempt to follow minimal residual disease (Billadeau et. al 1991;Billadeau et al, 1992; Billadeau et al., 1993; Chen and Epstein 1996;Cao et al. 1995). In all of these reports, the sequences of the PCRprimers were originally derived from amplification of bulk RNA or DNAisolated from tumor-containing material, which the present inventorsfind may amplify a sequence which is unrelated to the malignant clone.Another major problem with this approach is that the use of bulk nucleicacid to detect clonotypic sequence does not give quantitative results,and may grossly underestimate the frequency of clonotypic sequences.

A reliable method to quantitate malignant cells during assessment ofminimal residual disease does not currently exist. A reference by Yamedaet al., 1990 proposed a method based on cloning the products of a PCRamplification into bacteriophage and attempting an analysis of the ratioof phage carrying the clonotypic sequence to phage carrying any otherrearrangement. Billadeau et al. (1991) proposed to quantitate a PCRamplification of bulk nucleic acid by preparing a standard curve byseeding known numbers of clonotypic cells into a population ofnon-rearranged cells. Both proposed methods are very indirect. The FISHmethod of Cao, cited above, while it provides an analysis on a singlecell level, is too insensitive to be quantitative, measuring only thecells which express very high levels of mRNA.

In situ PCR and in situ RT-PCR are known in the art as means to detectnucleic acid sequences in single cells (U.S. Pat. No. 5,436,144 toStewart and Timm, 1995; Nuovo, 1994).

It is clinically important to be able to monitor the members of amalignant T or B lymphocyte clone, over time in order to determine theeffect of treatment on cells of malignant lymphocyte clones. Since mostof these malignancies are heterogeneous in differentiation state and/ormorphology, the only marker that unequivocally confirms a relationshipwith the malignancy is the immunoglobulin rearrangement of the IgH,light chain (k or l) or the T cell receptor a, b, g or d. Assays usingbulk nucleic acid from lymphocyte populations are not quantitative anddo not identify the clonotypic cell types present in blood, lymphoidtissue or bone marrow. Often the malignant lymphocyte comprising themajor cellular mass of primary tumor divide slowly or not at all, andmay be terminally differentiated with little generative capacity.Hematopoietic malignancies appear to be hierarchical with components atsequential states of differentiation not easily detected withconventional clinical assays, especially if they are present at lowfrequency. Thus at present, the effects of treatment on the fullhierarchy of malignant cells in diseases much as myeloma, lymphoma andchronic and acute lymphocytic leukemias, cannot be assessed.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the immunoglobulin heavy chain locus ingerm line and rearranged DNA.

FIG. 2 is a photograph illustrating the detection of amplified IgHrearrangements using consensus primers in single plasma cells and singleB cells from a multiple myeloma patient 6 months after chemotherapy.PBMC from patient JOD-5 were taken one month after completing 6 cyclesof chemotherapy. PBMC were stained with CD19-FITC and individual CD19+cells were deposited into lysis buffer in PCR tubes using the ELITEAutoclone cell deposition unit. DNA was amplified using hemi-nestedconsensus primers designed to detect IgH rearrangements. BMC werestained with CD38-PE and anti-human Ig-FITC and CD38+ cells with highforward and side scatter were sorted into PCR tubes. Lane 19 containedmolecular weight standards. Lane 1 contained a “water control” for thefirst step of the consensus PCR. Lanes 2-9: bands amplified from singleCD19+ PBMC. Lanes 11-18: bands amplified from single BM plasma cells.

FIG. 3 is a sequence alignment of CDR2 and CDR3 sequences obtained fromsorted blood B cells and autologous BM plasma cells. IgH bands amplifiedin hemi-nested PCR, shown in FIG. 2, were cut out, ligated into asequencing vector, and sequenced using dideoxy chain termination. (A)shows the CDR3 sequence obtained for 2 of the 10 sorted BM plasma cells(from FIGS. 2 and 3 others from the JOD-5 BM sample: in total, theproduct from 11 individual BM plasma cells was sequenced). Sequenceswere compared to known sequences using BLAST and the VBASE database, andaligned using GCG software. All 10 sequences were most closely relatedto the DP-31 VH3 gene family. The DP-31 sequence (line 1, SEQ ID NO.66)aligned with 2 representative JOD B cells (lines 2 and 3, SEQ ID NOS.67and 68 respectively) and 2 representative BM plasma cells (lines 4 and5, SEQ ID NOS.69 and 70 respectively), as well as the consensus JODsequence from the 10 plasma cell sequences (line 6, SEQ ID NO.71).Although the JOD sequence varies from the P-31 sequence in severalplaces, the same variation occurred in all JOD B and plasma cells, withfew exceptions (e.g. BM plasma cell 4.4, line 5, positions 213, 214,215). CDR2 and CDR3 sequences used for primers in PSA are underlined.(B) shows the JOD consensus sequence (line 1, SEQ ID NO.72) aligned withthe unrelated BM plasma cell 5.3 (line 2, SEQ ID NO.73) and with theunrelated B cell (line 3, SEQ ID NO.74), as well as a related BM plasmacell for comparison (line 4). The unrelated B cell sequence aligned withVH4a, and the unrelated plasma cell sequence aligned best, although atonly moderate homology, with the DP-31 sequence. The absence of bandsfrom water controls (FIG. 2) and the amplification of an unrelated IgHsequence from plasma cell #11, shows that the presence of a commonsequence for 10/11 cells amplifying a band, does not reflectcontamination by JOD DNA, and that amplification requires the presenceof a cell in the tube. The consensus rearrangement identified in 10/11plasma cells was designated as clonotypic for JOD.

FIG. 4 depicts patient-specific amplification (PSA) using CDR2 and CDR3primers specific for the JOD clonotypic VDJ rearrangement. Blood wastaken at 12 months post-diagnosis from patient JOD (JOD-6). DNA from 27single sorted B cells were amplified in a two step PCR. The initialamplification was carried out with consensus primers, followed by 40cycles of PSA PCR using primers homologous to the CDR2 and CDR3sequences (Table 1, SEQ ID NOS.8 and 9, respectively) of the JODclonotypic sequence identified in FIG. 3 (SEQ ID NO.72). The PSA step ofPCR was at high stringency (60¼C). (A) shows that a product of theexpected size was amplified from 9/27 individual B cells. (B) gives thesequence (SEQ ID NO.75) of the product amplified by the CDR2 and CDR3primers from 6 of the 9 B cells from which a patient-specific band wasamplified.

FIG. 5 shows the use of consensus primers in single cell RT-PCR toamplify IgH rearrangements from patient LAR BM plasma cells, thesequences obtained from the resulting PCR products, and the use ofpatient-specific primers derived from the sequence to amplify clonotypicsequences in single cells. (A) BMC from LAR, taken at diagnosis, werestained and individual plasma cells were sorted into PCR tubes. mRNA wasamplified at relatively low stringency using hemi-nested consensusRT-PCR. (B) The CDR2 to CDR3 sequence of LAR (SEQ ID NO.76). Bands from(A) were cut out and sequenced. For 3/3 BM plasma cells the same VDJsequence was obtained. (C) To confirm the number of single plasma cellsexpressing the sequence identified in (B) (SEQ ID NO.76), primershomologous to the patient-specific CDR2 and CDR3 regions were designed(Table 1) and used at high stringency to amplify mRNA from 48 individualBM plasma cells. 42/48 amplified a product of the expected size,confirming the sequence of (B) (SEQ ID NO.76) as clonotypic for LAR.

FIG. 6 demonstrates the specificity of LAR patient-specific primers.Clonotypic LAR sequences are amplified in bulk RT-PCR from unsorted LARBMC and from sorted LAR blood B cells but not from LAR blood T cells, orfrom BMC of unrelated patients. RNA was amplified from unsorted BMC,from sorted PBMC B cells and from sorted PBMC T cells usingpatient-specific CDR2/CDR3 primers (Table 1, (SEQ ID NO.8-11) at highstringency.

SUMMARY OF THE INVENTION

Recognizing the need for a clinically feasible test for assessingminimal residual disease in lymphoid malignancies which would require amore accurate, sensitive and quantitative method to detect clonotypicrearrangements in patient samples than was currently available, theobject of the invention was to develop a method with the followingfeatures: consistently accurate PCR primer specificity, a quantitativereadout, and high sensitivity. Ideally, the method should also becompatible with allowing qualitative identification of the cell typeswhich express a given clonotypic rearrangement.

The invention is based upon the discovery that the most accurate andquantitative information regarding the identity of a clonotypic sequenceand frequency of clonotypic cells in patient samples is obtained fromRT-PCR or PCR analysis of nucleic acid in whole cell lysates, ratherthan from purified bulk preparations of nucleic acid. The inventors madethe following observations:

PCR amplification of bulk nucleic acid preparations from MM plasmatumour cells using consensus primers for the VDJ region frequentlyyields a sequence which turns out not to be the clonotypic sequence ofthe malignancy.

Quantitative measurements of the frequency of a clonotypic sequence madeon bulk nucleic acid preparations tend to grossly underestimate thefrequency of such cells.

The inventors have circumvented these problems, which hinder thedevelopment of a clinical test for minimal residual disease:

(1) by performing RT-PCR or PCR directly in cell lysates made within afew hours of being harvested from the patient. In the case of MM patientsamples, PBMC or BMC are diluted in a small volume (several microliters)of an appropriate lyses buffer. RT-PCR or PCR is performed directly inthe cell lysate; and

(2) by performing RT-PCR in situ in intact cells. In situ RT-PCRamplification is carried out using patient specific VDJ region primersin cells affixed to slides, which allows for the direct visualizationand counting of clonotypic cells.

In accordance with the invention a method for the detection ofclonotypic gene rearrangements in patient cell samples comprises twophases. In the first phase, the nucleotide sequence of the clonotypicrearrangement is determined, and the patient-specific PCR probes aredesigned, using the following steps:

(a) isolating single cells or pools of up to 1000 cells by means of cellsorting, limiting dilution, or other means from a tumor cell-rich sampleof patient cells,

(b) amplifying a region of DNA comprising at least a portion of VDJ inseveral of the isolated single cells or pools o f up to 1000 cells byPCR or RT-PCR, using pairs of consensus framework primers which areknown to amplify DNA in the variable region of Ig or TCR, oralternatively, amplifying DNA known to contain another type of DNArearrangement, such as a chromosomal translocation using appropriateconservative primers;

(c) determining the nucleotide sequence of the amplified DNA segments;

(d) constructing patient-specific PCR primers, which bracket theamplified nucleotide sequence, specific for the CDR, CDR2, and/or theCDR3 regions, or the CDR1, CDR2, or CDR3 region, or any set of primersthat specifically amplify the unique hypervariable regions of the IgH, kor 1 immunoglobulin gene, or the TCR a, b, g or d chain, or any set ofprimers that specifically amplify a clonotypic rearrangement in lymphoidmalignancies.

(e) confirming that the patient-specific primers amplify the sequenceobtained in (c) above, and no other sequences.

The second phase of the method of patient specific amplificationcomprises the use of in situ RT-PCR or PCR to amplify a nucleotidesequence comprising a clonotypic rearrangement in intact cells, withoutremoving the DNA or RNA from the cell, as follows:

(a) performing in situ PCR or in situ RT-PCR, using the saidpatient-specific probes on patient tissue samples comprisingfractionated or unfractionated white blood cells, peripheral bloodmononuclear cells, or bone marrow cells, or any other patient samplessuch as tissue biopsies, which are either fixed to slides, or fixed insolution.

(b) directly detecting the resultant amplified DNA in cells, or, for anadditional confirmation of specificity, hybridizing the amplified DNA insitu to labeled nucleic acid probes comprising an internal portion(which excludes any primer sequence) of the clonotypic region.

An alternative or complement to the second phase of the method describedabove comprises determining the frequency of clonotypic cells by meansof a limiting dilution RT-PCR or PCR assay as follows:

(a) diluting cells in series to give from approximately 1000 cells to 1cell per tube,

(b) lysing the cells in the tubes,

(c) performing RT-PCR or PCR on the resulting material,

(d) counting the number of tubes containing the clonotypic sequence foreach dilution, and

(e) estimating the frequency of clonotypic cells.

The advantage of the limiting dilution methodology is that a clonotypicsequence can be detected from a single cell as in the in situ assay, butthe limiting dilution method is simpler and more cost effective. Cellsdestined for this method can be processed quickly and stored at −80° C.for later analysis. Cells destined for in situ RT-PCR must be fixed,washed and applied to slides within 18-24 h.

The invention extends to a patient-specific kit designed for theanalysis of clonotypic cells in patient samples comprising at least onebut preferably two patient-specific PCR primers, and a nucleic acidprobe comprising at least a portion of the clonotypic V(D)J region whichis amplified by the patient-specific PCR primers or the patient-specificprimer and an appropriate consensus primer.

The method is used to detect malignant lymphocytes in patient samples ina wide variety of applications, which include, but are not limited tothe following:

to monitor clonotypic cells before during and after treatment in anylymphoid malignancy in which any Ig or any TCR genes are rearranged,including, but not limited to multiple myeloma, Hodgkin's lymphoma, andALL;

to monitor clonotypic cells before, during or after treatment inlymphoid malignancies in which the clonotypic rearrangement comprises achromosomal translocation;

to monitor the presence of clonotypic cells in a population of cells tobe transplanted, for example bone marrow cells or isolated stem cells;

to monitor the presence of clonotypic cells in pre-malignant conditionssuch as monoclonal gammopathy of undetermined significance, indolentmyeloma, or smoldering myeloma;

to monitor the presence of clonotypic cells in autoimmune diseasescharacterized by autoimmune clonal expansion;

for use in the identification of the variety of cell types representingthe various differentiation stages which comprise a malignant clone; and

for use in the development of treatment protocols which requiresensitive tests for malignant cells in blood, bone marrow and othertissues.

Broadly speaking, one aspect of the invention is a method fordetermining the correct patient-specific clonotypic nucleic acidsequence for a tumour comprising:

performing RT-PCR or PCR with consensus primers using the unpurifiednucleic acid released from small numbers of lysed tumour cells, andpreferably a single tumour cell, as a template, to generate a product,and

obtaining the nucleotide sequence of the product.

Another aspect of the invention is a method for analyzing the number ofcells in a population of cells which contain a clonotypic sequence,comprising:

performing RT-PCR or PCR with at least one correct patient specificclonotypic primer to amplify the clonotypic nucleotide sequence inintact cells, and

detecting the amplified clonotypic sequence in the intact cells.

A further aspect of the invention is a method for analyzing the numberof cells in a population of cells which contain a clonotypic sequencecomprising:

performing RT-PCR or PCR with at least one correct patient specificclonotypic primer to amplify the clonotypic nucleotide sequence usingthe unpurified nucleic acid released from cells which have beensubjected to limiting dilution and lysed, and

detecting the amplified clonotypic sequence.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises methods for the detection of clonotypic DNArearrangements in lymphoid tissues. The inventors encountered twoproblems during their investigation of the role of blood-borneclonotypic B cells in myeloma. The first was that no existing method ofscreening for clonotypic rearrangements was both sensitive enough andquantitative enough to provide a reliable count all of the clonotypiccells in all states of differentiation. The second was that inattempting to generate patient-specific PCR primers using bulk RNA orDNA isolated from myeloma bone marrow cells, it became evident thatconsensus primers to the IgH variable region sometimes amplify a VDJsequence which is not the clonotypic rearrangement present in themyeloma cells. The reason for this was not clear. However, use of thewrong PCR primers is fatal to any attempt to assay for clonotypic cellsin patient samples.

Identification of Clonotypic Sequence, Design and Testing ofPatient-specific Primers

One aspect of the invention provides for a reliable method to generatePCR primers for use in screening. This aspect of the invention is basedon the premise that in order to unequivocally determine the rearrangedIg or TCR sequence of a malignant clone, it is necessary to amplify thisrearrangement from a small number of cells, preferably individual cells,sequence the amplified product, and then confirm that the majority ofcells thought to be malignant actually express the identified sequence.This aspect of the invention comprises the following steps:

(a) performing PCR using consensus framework primers on a number ofsingle tumor cells or groups of 1000 or fewer tumor cells to amplify theclonotypic rearrangement,

(b) obtaining the amplified PCR products by gel electrophoresis,

(c) obtaining the nucleotide sequence of the amplified PCR products fromseveral tumor cells before assigning a sequence to the malignantclonotypic rearrangement,

(d) preparing patient specific DNA primers based on the clonotypicsequence,

(e) testing the patient-specific primers, once constructed, for theirability to amplify the clonotypic sequence and only the clonotypicsequence in patient samples.

This aspect of the invention is directed toward overcoming the problemthat use of homogenized nucleic acid as the initial source of thesequence can amplify an infrequent and thus inaccurate sequence. Ittherefore provides for the identification of the clonotypicrearrangement, using single tumor cells, or very few tumor cells, ratherthan bulk homogenized DNA. For this procedure, it is desirable to obtaina tumor-rich sample of cells, or to enrich the cell sample for tumorcells, if possible. For multiple myeloma, bone marrow plasma cells arean ideal source of tumor material. Single cells can be sorted using acell sorter such as the ELITE Autoclone (Coulter Electronics), usingsurface markers (which are detectable by fluorescently labeledantibodies), and/or size gating to distinguish tumor cells fromnon-tumor cells in the patient samples. For example, a large size, and ahigh concentration of both the CD38 marker (detectable by an antibody toCD38) and immunoglobulin (detectable by an antibody to immunoglobulin)are diagnostic for myeloma plasma cells, as detailed in the Materialsand Methods section below. If the patient cell sample contains primarilytumor cells, the single cells can be plated by limiting dilution,without the need for cell sorting.

The single cells, prepared as indicated above, are placed into a smallvolume consisting of a few microliters of an appropriate lysis buffer,depending on whether RNA (RT-PCR) or DNA (PCR) will be used as thestarting material for amplification (see Materials and Methods). In allcases control samples which do not contain a cell are included. At thispoint it is possible to amplify DNA (which is present in only one copy)in the single cell, or to copy RNA (which may be present in hundreds ofcopies, or none at all, depending on the cell type) into cDNA which isthen amplified. In cells such as myeloma plasma cells, RNA is relativelyabundant, making RT-PCR practical. However, RNA is much more labile thanDNA, so cell samples which are not optimally fresh, in which RNA may bedegraded, could still be used for PCR, rather than RT-PCR. If RT-PCR isdone, then RNA is copied into cDNA by means of the enzyme known asreverse transcriptase, using methodology well known in the art. Themethodology used by the inventors for this step is outlined in theMaterials and Methods.

PCR is then carried out in order to amplify all sequences with VDJrearrangements. The appropriate consensus framework primers are chosenbased upon known sequences in the constant or framework regions of theIg or TCR gene which bracket the VDJ recombination sites, or a chosenhypervariable sequence. For Ig, the rearranged heavy chain has morediversity than the rearranged light chain, so that it is a better choiceas a probe for clonotypic sequence. However, if the heavy chain is notrearranged in the tumor being assayed, primers to the light chains canbe used .

In order to minimize spurious amplification products, the inventors in apreferred embodiment carried out two rounds of PCR using hemi-nestedconsensus framework primers specific for IgH, the sequences of which aregiven in Table 1, (SEQ ID NOS.5-7). The first round of amplification wasdone with an upstream primer which hybridizes in the FR2 region (FR2,SEQ ID NO.5), and a downstream primer which hybridizes in the J region(JH1, SEQ ID NO.6). An aliquot of the material from the first round ofamplification was used for a second round of amplification using thesame upstream primer, and a downstream primer (JH2, SEQ ID NO.7) whichhybridizes in the J region, just upstream (on the 5′ side) of the JH1primer.

The products of the second round of amplification are subjected toelectrophoresis in an agarose or polyacrylamide gel, using methodologywhich is well known in the art, including the use of appropriate sizemarkers. The PCR products are variable in length, depending on thechoice of primers and length of the DNA segment which is bracketed bythe primers. However, most products will be within the range of 150-200nucleotides. The bands are cut out of the gel, separated from the gelmaterial using methodology well known in the art, ligated into asequencing vector and sequenced using standard methodology (seeMaterials and Methods). The sequences obtained for the panel of singlecells can be conveniently compared to known Ig, TCR or other sequencesusing information contained in data bases such as BLAST and the VBASEdatabase, and can be aligned using appropriate software. Generally, thesequence obtained aligns with one of the VH families if IgH PCR primershave been used. Once the sequences of a number of cells are obtained andaligned, it is then possible to assign a consensus sequence to theclonotypic rearrangement. FIG. 3A exemplifies the alignment ofsequences, and the consensus sequence assigned in a preferredembodiment. The experience of the inventors in using single purifiedmyeloma plasma cells as starting material is that many of the sequencesobtained are identical or nearly identical. However, the number ofsequences obtained which are clearly unrelated to the malignant clonewill vary with the purity of the tumor cell starting material.

Once the clonotypic sequence is known, it is possible to designpatient-specific PCR primers specific for one or more of the mostvariable regions in the sequence. In a preferred embodiment, theinventors chose the upstream primer in the CDR2 region and thedownstream primer in the CDR3 region of the rearranged IgH.

The next step is to confirm that the primers are truly specific, in thatthey amplify the clonotypic sequence that they bracket, and only thatsequence . Single cells from the tumor cell-rich source are again usedas starting material for PCR or RT-PCR. Appropriate controls, such asunrelated single B cells or T cells are also included in the experiment.The first round of amplification is carried out using the consensusprimers (in the preferred embodiment, the FR2 and the JH1 primers).However, the second round of amplification is performed using thepatient-specific primers. Products are run on a gel and sequenced asabove. If the primers amplify the correct product—the clonotypicsequence in tumor cells, but do not amplify any product in irrelevantcells, then they have been vetted as patient-specific primers suitablefor use in analytical patient-specific amplification (PSA), to bedescribed below. If the primers do not amplify a product, or if theyamplify a spurious product, other primers based on the clonotypicsequence can be selected.

Analysis of Clonotypic Cells Using PSA in Single Cells

In order for a screening test to be useful clinically for the purpose offollowing the progress of disease and treatment, and in order forclinical decisions to be based upon the results of the test, it musthave both a quantitative readout (the assay must be done at the level ofsingle cells, rather than bulk preparations of nucleic acid made from awhole population of cells), and sensitivity (the RNA or DNA reflectingthe clonotypic rearrangement in the single cells must be amplified todetectable levels, so that clonotypic cells expressing low levels of RNAor no RNA can be detected).

A further aspect of the invention therefore provides for the use of insitu PCR or RT-PCR with patient-specific PCR primers, which may beproduced as indicated above, to detect clonotypic rearrangements insingle cells. In a preferred embodiment, a novel use for in situ RT-PCRis described for amplifying the CDR1-CDR2-CDR3 regions of individualcells placed on a slide, or suspended in a solution. A cell with atleast one gene copy of the patient-specific rearrangement is amplifiedand is visualized using an appropriate means, which may be colorimetryor autoradiography if the process is carried out on slides, orfluorescence if the assay is carried out in suspension.

The patient samples to be analyzed can include unpurified white cellsfrom blood after red cell lysis (WBC), isolated peripheral bloodmononuclear cells (PBMC), or bone marrow mononuclear cells (BMMC). Anyof these cell populations can be subjected to further fractionation,selection or depletion using standard methods such as FACS sorting. Forexample, to quantitate the proportion of T cells with a specificclonotypic rearrangement, one approach would be to positively select forT cells using an antibody to a T cell marker such as CD3 using a cellsorter, and to examine the selected T cells for the presence of theclonotypic rearrangement. Cells are generally formalin fixed beforesorting and purification in order to preserve RNA. If sorted cells areto be used, approximately 10,000 cells (using an ELITE Autoclone(Coulter) or comparable equipment) sorted into microtitre wells aresufficient. The method is also amenable to using tissue biopsies orsections from solid organs such as spleen and lymph node.

For analysis on slides, the cells can be placed onto appropriatelytreated siliconized glass slides (for example, In situ PCR glass slidesfrom Perkin Elmer) in three spots each, to accommodate processing ofpositive and negative controls on the same slide. RT-PCR is carried outessentially according to published methods (Nuovo, 1994), afterfine-tuning the system for the particular cell types and PCR primersbeing used. The conditions for PCR and RT-PCR which gave optimal resultsfor the primers used in the Examples are detailed in the Materials andMethods.

Another aspect of the invention encompasses, as an alternative toamplification of clonotypic rearrangements in cells affixed to slides,performing the in situ PCR or in situ RT-PCR in cells in suspension,using methods generally disclosed in U.S. Pat. No. 5,436,144 to Stewartand Timm (1995). Detection of clonotypic cells is accomplished by meansof flow cytometry. For example, the PCR amplification step can becarried out in the presence of a biotinylated dNTP, which can bedetected in flow cytometry using fluoresceinated (FITC-labeled) orphycoerytherin-labeled avidin. Using this methodology, it is feasible touse two-color flow cytometry to detect both the clonotypic DNA markerand a cell surface marker. It is therefore possible to positively andsensitively identify malignant cells which exist at a particulardifferentiation state, as defined by expression of cell surface markers,such as CD10, CD34, or CD38.

In a further aspect of the invention, the clonotypic specificity of theproduct of in situ amplification is confirmed by a step comprisinghybridization of this product to a nucleic acid probe comprising aninternal portion (excluding any primer sequence) of the clonotypicregion (for example the IgH V(D)J region). The amplification step iscarried out in the absence of labeled dNTPs, so that the PCR product isunlabeled, and detection of cells positive for the clonotypic sequenceis done by means of a labeled nucleic acid probe. Alternatively, PCRproducts could be labeled with a label which will not interfere withdetection of the hybridization probe. The process of in situhybridization, both on cells fixed to slides and in cells in suspension,is well known in the art. The nucleic acid probes consist of a labeledsingle stranded RNA.

In a further aspect of the Invention, rather than amplifying theclonotypic sequence in situ in intact cells, the cells are subjected tolimiting dilution, down to 1 cell per tube, lysed, and RT-PCR or PCR iscarried out on the unpurified nucleic acid which has been released fromthe cells. This method differs from methods used in the prior art inthat the released nucleic acid is used without further purification oreven ethanol precipitation. The inventors have found that by performingRT-PCR on unmanipulated cell lysates, it is possible to detectclonotypic sequence which is the product of a single cell. The inventorsbelieve that when nucleic acid, especially RNA is purified, material islost or degraded. This can lead to a gross underestimation of the numberof cells within a population which contain clonotypic sequence. Theinventors avoid loss and degradation of RNA in cell samples by firstly,placing cells in a buffered solution which inhibits degradation of RNA,and secondly, by not performing any manipulations on the RNA before itis subjected to reverse transcription and PCR.

This aspect of the invention, which is capable of detecting single cellscontaining clonotypic sequence, provides an alternative to the use of insitu RT-PCR and in situ PCR. It is therefore possible to use this methodto determine the frequency of clonotypic cells in a population of MMPBMC, MM BM or other cells. A limiting dilution of cells, followed bypatient specific amplification is performed. Defined numbers of PBMC orother cells are placed in PCR tubes in a dilution series to give fromapproximately 1000 cells to 1 cell per tube as soon as possible afterisolation. Controls containing no cells are included. The products ofamplification are detected on ethidium bromide stained gels.

Generally, with MM PBMC, all wells containing 1000, 300, 100 and 30cells contain the clonotypic nucleotide sequence, indicating that atleast 3 out of 30 or 10% of cells are clonotypic. If 2 wells containing1 cell per well are positive, that would indicate that approximately 66%of PBMC are clonotypic. More than 3 replicates can be used to obtainmore precise numbers.

The details of this aspect of the invention are outlined in Example 3below.

The inventors anticipate that this aspect of the invention will allowfor testing of patient samples which are obtained in centres which arenot equipped to handle in situ RT-PCR or even cell sorting. Theinventors envision that a clinical lab would obtain a “sample collectionkit” comprising tested reagents and tubes. For example:

PCR microtubes containing 8 ml of lysis buffer (1.2×transcriptionbuffer, a non-ionic detergent and RNASE inhibitor),

PBS,

tubes for diluting cells, and

instructions.

The instructions would direct the technician to:

process blood or bone marrow on Ficoll to obtain mononuclear cells,which is a standard laboratory procedure;

re-suspend the washed cells to a concentration of one million (10⁶)cells per ml of PBS;

make dilutions (3 or 10 fold) by diluting the appropriate volume of cellsuspension into PBS to generate dilutions down to 10³ cells/ml (at thatdilution 1 microliter contains 1 cell) (for example, 100 ml of 10⁶cells/ml into 900 microliters of PBS generates a dilution containing 10⁵cells/ml, etc.);

transfer 1 microliter from each cell suspension into ice cold PCR tubescontaining the lysis buffer in triplicate; and

freeze at −80° C., and ship on dry ice to a core lab.

In the core lab, bone marrow cells samples treated as above would besubjected to RT-PCR using consensus primers to determine the clonotypicsequence for the patient according to the method of the invention.Blood, bone marrow or other types of cell samples treated as above wouldbe subjected to RT-PCR using patient specific primers to determine thefrequency of clonotypic cells in the samples.

Applications

The methods of the invention can be used for the analysis of anymalignancy which carries a clonotypic rearrangement for which it ispossible to generate patient-specific PCR primers. These include:

(1) malignancies of the T cell lineage, which carry rearrangements ofthe genes for the a, b, g or d chains;

(2) malignancies of the B cell lineage, which carry rearrangements ofthe genes for the immunoglobulin heavy chain, or the k or l lightchains,

(3) hematological malignancies in which a chromosomal translocationprovides a clonotypic marker, many of such translocations involve an Igor TCR locus (exemplified by, but not limited to, translocationsinvolving chromosome 11 band q23, which occurs frequently in bothmyeloid and lymphoblastic leukemias (Rowley, 1990), and thetranslocation of the c-myc protooncogene with the IgH locus (Taub et.al. 1982). Translocations, involving Ig or TCR loci have been identifiedin stem cell leukemia, T cell-ALL, T cell CLL, Adult T cell Leukemia,T-prolymphocytic leukemia, high and low grade lymphoma, diffuselymphoma, B cell CLL, multiple myeloma, follicular lymphoma, B-CLL, aswell as Burkitt's lymphoma.

The methods of the invention can be used to detect malignant lymphocytesin patient samples for a wide variety of applications:

to monitor clonotypic cells before during and after treatment in anylymphoid malignancy;

to monitor the presence of clonotypic cells in a population of cells tobe transplanted, for example bone marrow cells or isolated stem cells;(The failure of treating malignancies such as multiple myeloma withablative chemotherapy and radiotherapy followed by autologous bonemarrow or stern cell transplantation has been attributed in part tocontamination of the transplant with cryptic tumor cells.)

to monitor the presence of clonotypic cells in pre-malignant conditionssuch as monoclonal gammopathy of undetermined significance, indolentmyeloma, or smoldering myeloma;

to monitor the presence of clonotypic cells in autoimmune diseasescharacterized by autoimmune clonal expansion;

for use in the identification and quantitation of the variety of celltypes representing the various differentiation stages which comprise amalignant clone; and

for use in the development of treatment protocols which requiresensitive tests for malignant cells in blood, bone marrow and othertissues.

The invention can be better understood by reference to the followingnon-limiting examples, which illustrate the use of the methods of theinvention to quantitate the number of clonotypic B cells in the blood ofmultiple myeloma (MM) patients over time, before, during and afterchemotherapy.

MM is characterized by the presence of monoclonal immunoglobulin in theblood, lytic bone lesions, and often large numbers of monoclonal plasmacells in the bone marrow. Although many patients respond to treatment,nearly all relapse and become refractory to treatment (Barlogie et al.,1989; Greipp, 1992). While it is clear that monoclonal plasma cellslocated in the bone marrow directly or indirectly mediate most symptomsof myeloma, these cells do not appear to have the qualities of growthand spread required of a malignant progenitor cell. Consistent withthis, the degree of reduction of plasma cell burden (Bergsagel, 1979) orof monoclonal immunoglobulin (Palmer et al., 1989) does not correlatewith enhanced survival, and the extent to which bone marrow used fortransplantation is contaminated with plasma cells has little impact onpatient survival (Barlogie et al., 1989). A number of observations haveled to the view that the generative compartment in myeloma includes Blineage cells found in the bone marrow, the blood or both, at a stage ofdifferentiation preceding that of plasma cells (Pilarski and Jensen,1992; Bergsagel et al. 1995; Pilarski et al, 1996; Jensen et al., 1991;Boccadoro et al, 1983; Hulin et al, 1978; Omede et al, 1993;Caligaris-Cappio et al, 1985; Berenson et al, 1987; Billadau et al.,1993; Takashita et al, 1994). Normal plasma cells are terminallydifferentiated B cells which do not divide, but which are active inimmunoglobulin synthesis. There is no direct evidence that the malignantclonotypic plasma cells in multiple myeloma divide either. However,because there has been no way to detect the generative precursors ofmultiple myeloma plasma cells, clinical analysis as well as treatmenthas focussed on the plasma cell. Precursors have thus far beenoverlooked, although their presence or absence may be crucial todesigning successful treatment protocols.

A number of studies have demonstrated cells in blood of myeloma patientswith an IgH rearrangement identical to that of autologous bone marrowplasma cells (Bergsagel et al, 1995; Bersenson et al, 1987; Takashita etal, 1994; Bakkus et al, 1994; Billadeau et al, 1992; Corradini et al,1993; Gazitt et al., 1994; Owen et al., 1994; Sassel et al., 1990;Dreyfus et al., 1993; Mariette et al., 1994; Cirradini et al., 1995;Chen and Epstein, 1996). The differentiation stage of these blood cellsand their number has been controversial. A first step towards evaluatingthe extent to which peripheral blood lymphocytes include malignantmyeloma relatives is to quantitate the number of clonotypic B cells inthe circulation. Previous work showing the presence in circulating cellsof clonotypic rearrangements have provided a wide range of estimates(Billadeau et al., 1992; Dreyfus et al., 1993; Chen and Epstein, 1996;Vescio et al., 1995; Billadeau et al., 1995). All of these estimates arebased on the apparent frequency of a given sequence within homogenizedDNA or RNA from a heterogeneous population of cells.

The inventors have previously identified a large subset of cells bearingCD19+ (a diagnostic marker for B cells) in the blood of myeloma patientssome of which have clonotypic sequences (Bersagel et al., 1995), withthe phenotype of late stage B cells and properties consistent withmalignant status (Pilarski et al. 1996). In the examples below, theinventors used the methods of the invention to unequivocally determinethe clonotypic sequence, and to quantitate the number of clonotypic CD19+ B cells in the blood of multiple patients.

Materials and Methods

Patients: Blood and bone marrow were obtained after informed consentfrom 20 patients with multiple myeloma, at diagnosis, duringintermittent chemotherapy and after treatment. Samples are numberedsequentially, e.g. JOD-1, JOD-6, etc. Peripheral blood was drawn intoheparinized tubes and purified over Ficoll Paque (Pharmacia, Dorval QB)as previously described (Bergsagel et al., 1995) to give peripheralblood mononuclear cells (PBMC). Bone marrow cells (BMC) were alsopurified using Ficoll Paque. All samples were purified immediately afterbeing drawn, and were stained for cell sorting (as outlined below) andfixed within 4 hours after collection, to preserve mRNA. Samples for insitu RT-PCR were stored for up to 24 hours in fixative prior to sorting.For single cell PCR and RT-PCR, all samples remained unfixed, werestained, sorted and processed within 4 hours post-collection of thesample.

Antibodies and reagents: FMC 63 (CD19; a diagnostic marker for B cells;Pietersz et al, 1995; Zola et al., 1991) was conjugated to FITC. Leu-4PE(CD3; a diagnostic marker for T cells) and Leu17-FITC (CD38; adiagnostic marker for plasma cells) were from Becton Dickinson (SanJose, Calif.). Ig2a-PE, IgG1 and goat anti-mouse Ig-PE were fromSouthern Biotech (Birmingham, Ala.). Anti-human Ig F(ab)2 fragmentscoupled to PE and F(ab)2 fragments of goat-anti-mouse PE were fromSouthern Biotech.

Immunoflourescence (IF) and cell sorting: Staining for surface phenotypeutilized 1 or 2 color IF with CD19-FITC and CD3-PE, as described inPilarski and Belch, 1994 and Bergsagel et al., 1995. All experimentsincluded controls with isotype matched monoclonal antibodies. CD19+ andCD3+ subsets of PBMC were sorted using the ELITE (Coulter, Hialieh,Fla.). BMC were stained with CD38-FITC and anti-human Ig-PE followed bysorting of the cells with high forward and side scatter that werestained by both CD38 and Ig reagents. Sort gates were set to includeonly those cells with staining brighter than the relevant isotypecontrols, as previously described (Bergsagel et al., 1995). For singlecell experiments, individual CD19+ PBMC or CD38^(hi) large BMC weresorted into individual wells of a microtitre plate, or directly into 0.2ml thin walled PCR tubes. On reanalysis, sorted CD19+ populations had apurity of 95% or greater for the defining phenotype. PBMC had nodetectable contamination with any peripheral plasma cells as defined bytheir relatively low cytoplasmic Ig content (Bergsagel et al., 1995),and the absence of morphologically identifiable plasma cells incytospins of sorted subsets, in cytospins of PBMC or in smears ofpatient blood. To avoid any contamination between samples, and inparticular to avoid contamination of blood cells with BM cells, bloodsamples were always sorted prior to bone marrow samples, and tubing inthe flow cytometer was always washed with bleach between sorts.

After Ficoll partial purification of BMC, the BMC can be furtherseparated into B or T cells or plasma cells using antibody coatedcolumns and these cells. Any of these populations of cells could bediluted using limiting dilutions to yield one cell per well and thisprocedure could be used in place of flow cytometry.

Morphology of sorted CD19+ MM PBMC: Sorted CD19+ PBMC were place onslides, and were stained with Wright's stain. Slides were examinedmicroscopically for morphological characteristics.

Patient-specific amplification (PSA): For amplification ofpatient-specific sequences, primers to CDR2 and CDR3 regions of therearranged IgH VDJ from individual BM plasma cells were designed andused for in situ RT-PCR. This was found to be more specific than was theuse of a CDR3 primer paired with a consensus FR2 primer. PSA utilized aprimer from the 5′ terminus of the CDR2 region paired with a primer tothe entire CDR3 region. The sequences for patients JOD and LAR PSAprimers are given in Table 1 (SEQ ID NOS.8-11). Primer sequences weredesigned based on the IgH VDJ sequence present in the majority ofindividual BM plasma cells. For all patients, the CDR2/CDR3amplification was done using autologous T cells as a negative control,and the specificity of the amplification was confirmed by testing theprimers on B cells from an unrelated patient. For JOD, the sequencesused in PSA were detected in DNA from 10/11 single BM plasma cells. ForLAR, the presence of the clonotypic sequence was confirmed in the mRNAfrom 42/48 individual sorted LAR BM plasma cells using single cellRT-PCR with patient-specific CDR2/CDR3 PSA giving a product of theexpected size, 162 base pairs.

Single cell PCR: Single cells were sorted into 0.2 ml PCR tubesassembled on a Micro Amp base (Perkin Elmer, Mississauga, ON). Each ofthe tubes contained 5 ml of lysis solution (200 mM KOH, 50 mM DTT).After sorting, tubes were incubated in a thermal cycler (Perkin Elmer)for 10 minutes at 65° C. followed by addition of 5 ml of neutralizingsolution (900 mM Tris-HCl pH 9.0, 300 mM KCl, 200 mM HCl). Next, 40 mlof a PCR mix (0.1 mM dNTPs [Boehringer Mannheim, Laval QB], 10 mMTris-HCl pH9.0, 0.1% Triton-X-100, 2 mM MgCl2, and 5 units of TAQpolymerase [Gibco/BRL, Burlington ON] containing 0.01 mM of both FR2 andJH1 primers [see Table 1], was added to each sample. Samples were cycledas follows: 180 seconds at 95° C. (initial denaturation step), followedby 40 cycles of 30 seconds at 94° C., 30 seconds at 52° C., and 60seconds at 72° C., followed by a 10 minute terminal incubation at 72° C.For consensus amplification, two ml of each sample was then transferredinto a second PCR tube containing 48 ml of a PCR mix as above containing0.01 mM of both FR2 and JH2 primers, and cycled as above. Forpatient-specific amplification (PSA) with single cells, the second roundof amplification was performed as above, except with PCR mix containing0.01 mM of both CDR2 and CDR3 primers instead of the consensus FR2 andJH2 primers, and a higher stringency annealing temperature of 60° C.,instead of 52° C. The final products were analyzed by electrophoresisthrough 6% polyacrylamide gels or on 2% agarose gels in 0.5×Tris/BoricAcid/EDTA (TBE) buffer (Sambrook et al., 1989), followed by ethidiumbromide staining of the gels, and visualization of bands under UV light.

Single cell RT-PCR: Single cells were sorted into 0.2 ml PCR tubes asfor single cell PCR, above. Each tube contained 4 ml of RT-Lysissolution (SuperScript first strand buffer from Gibco/BRL [0.25 MTris-HCl (pH 8.3), 0.37 M KCl and 15 mM MgCl₂], 0.5% NP-40, 0.01 M DTT,0.25 mM dNTPs, 200 units of RNAse inhibitor, and 0.006 mMdT16 (auniversal poly dT primer). After sorting, the samples were heated to 70°C. for 10 minutes, placed on ice, and 1 ml (10 units) of reversetranscriptase (SuperScript, Gibco/BRL) was added to each tube. The tubeswere incubated at 42° C. for 30 minutes, and the reactions were stoppedby heating at 99¼C for 3 minutes. Two ml of synthesized cDNA weretransferred into fresh PCR tubes containing 48 ml of PCR mix (0.1 mMdNTPs, 10 mM Tris-HCl pH 8.3, 2 mM MgCl2, 2 units of TAQ polymerase)containing 0.01 mM each of FR2 and JH1 primers. Samples were heated for3 minutes at 95° C., followed by 25 cycles of 30 seconds at 94° C., 30seconds at 52° C. and 1 minute at 72° C. For consensus PCR, 2 ml of thisPCR-amplified mixture were transferred into a third tube containing 48ml of the PCR mix containing 0.01 mM of both FR2 and JH2 primers, andcycled as above for 25 cycles. For PSA, this final amplification wasperformed as above, except with patient-specific CDR2 and CDR3 primersand an annealing temperature of 60° C. The final products were analyzedby electrophoresis through 2% agarose gels in 0.5×TBE buffer, followedby ethidium bromide staining of the gels, and visualization of bandsunder UV light.

In situ RT-PCR: In situ reverse transcriptase polymerase chain reaction(RT-PCR) (Nuovo, 1994) was used to quantitate the proportion of sortedPBMC expressing IgH mRNA, CD19 mRNA and clonotypic VDJ rearrangements.PBMC from MM patients were stained in double direct immunofluorescencewith monoclonal antibody to CD19 (CD19-FITC) and to T cells (CD3-PE),and fixed in 10% formalin/PBS overnight. Using the ELITE Autoclone(Coulter), each PBMC sample was sorted at 10,000 cells per well of aflat bottom 96 well microtitre tray into T (CD3+19−) and B (CD3-19+)fractions. For some samples the whole blood lysis method (BectonDickinson) was used to prepare cells for the in situ RT-PCR, as well asunfractionated PBMC. Rapid processing prior to the fixation step wasessential to preserve mRNA. Samples were placed in 3 spots, at 10,000cells per spot, on In situ PCR glass slides (Perkin Elmer) and airdried. Cells were permeabilized using 2 mg pepsin (Boehringer Mannheim)per ml of 0.01N HCl. The time of pepsin digestion was carefullyoptimized. Pepsin was inactivated to a 1 minute wash in DEPC(diethylpyrocarbonate)-treated water, followed by a 1 minute wash in100% ethanol. Digestion with 1000 U/ml of DNAsel (RNAse-free, BoehringerMannheim) removed genomic DNA prior to reverse transcription. Incubationof the sample with DNAseI was performed in the In situ PCR System(Perkin Elmer) thermal cycler at 37° C. overnight. DNAseI was removed bya 1 minute wash in DEPC-treated water followed by a 1 minute wash in100% ethanol. In situ reverse transcription was performed to 60 minutesat 37° C. only for the test samples under standard conditionsrecommended by the manufacturer using SuperScript (Gibco/BRL) and theuniversal primer, dT16. After washing with water and ethanol, an In situCore Kit (Perkin Elmer) was used to amplify a target sequence during25-30 cycles (94° C. for 1′, 56° C. for 1′ and 72° C. for 1.5′) with adirect incorporation of DIG-11-dUTP (Boehringer Mannheim) during PCR tolabel the product. Amplified DNA was detected using anti-DIG Fabconjugated with alkaline phosphatase (Boehringer Mannheim), followed byincubation with NBT/BCIP substrate solution vitro blue tetrazoliumchloride/5-Bromo4-chloro-3indolyl-phosphate, 4-toluidine salt,Boehringer Mannheim). Color development was monitored under themicroscope. Negative controls for every sample included omitting the RTstep to confirm digestion of genomic DNA which would otherwise lead toamplification of non-specific PCR products. As a positive control, mRNAfor a housekeeping gene, histone, was amplified to quantitate the numberof cells on the slide with intact mRNA. As a control for the specificityof the primers used to amplify IgH mRNA and CD19, autologous T cellswere tested and were negative for both IgH and CD19 mRNA, as expected.Primer pairs are given in Table 1. IgH mRNA was detected using consensusprimers to FR2 (SEQ ID NO.5) and JH (SEQ ID NOS.6 and 7).

RT-PCR using bulk RNA: RNA was prepared from 0.1-10×10⁶ unfractionatedBMC, PBMC or sorted populations of B and T cells of the same patientusing Trizol (Gibco/BRL) according to manufacturer's directions. (The Tcells, collected at the same time as B cells in a doubleimmunofluorescence sort, serve as a negative control.) Afterpurification, 1 microgram of RNA was reverse transcribed usingSuperScript reverse transcriptase (Gibco/BRL) and the universal primeroligo dT₁₅ using manufacturers instructions. Briefly, RNA was incubatedwith the primer for 10′ at 70° C., chilled on ice and 5×First StrandBuffer (Gibco/BRL), 0.1M DTT, 0.25 mM dNTPs and 200U SuperScript reversetranscriptase were added. The reaction tube was placed at 42° C. for30′, followed by heating for 3′ at 100° C. PCR was performed understandard conditions. Briefly, 2 ml cDNA from the reverse transcriptasereaction was added to 48 ml of PCR Buffer (Gibco/BRL), containing 2 mMMgCl₂, FR2 and JH1 primers as described above for single cell RT-PCR,and 1U TAQ polymerase. Samples were cycled on the Perkin Elmer ThermalCycler 9600 for 25 cycles of 30 seconds at 94° C., 30 seconds at 50° C.and 45 seconds at 72° C. For consensus RT-PCR, a second round ofamplification was carried out using FR2 and JH2 primers, and cycling asabove. For PSA, the second round of amplification utilized patientspecific primers CDR2 and CDR3 for 25 cycles at an annealing temperatureof 60° C. The PCR products were analyzed by electrophoresis on 2%agarose gels in TBE buffer. The gels were stained with ethidium bromide,and bands were visualized with UV light.

DNA Sequencing: Sequencing was performed using techniques well known inthe art, with a³²P-dCTP (Amersham, Oakville ON) using either theSequenase 2.0 system (Amersham) or the cycle sequencing kit (PerkinElmer) following the manufacturer's instructions. For patient JOD, theFR2/JH2 PCR product was subcloned into a Bluescript vector expressed inthe DH5 strain of Escherichia coli, purified, and sequenced usinguniversal sequencing primers. For LAR, the FR2/JH2 product was digestedfrom the low melting point gel using b-Agarase (New England Biolabs,Mississauga ON) according to the manufacturers directions, and sequenceddirectly using the FR2 and JH2 primers. The products of sequencing wereanalyzed and aligned using GCG and NCBI BLAST programs.

EXAMPLE 1 PATIENT JOD

Amplification of Rearranged IgH Sequences in MM PBMC CD19+ Cells andBone Marrow Plasma Cells Using IgH Consensus Primers.

Substantial numbers (mean=28-33% (Bergsagel et al, 1995)) of CD19+ PBMCare detectable in PBMC of multiple myeloma patients (Pilarski andJensen, 1992; Bersagel et al., 1995; Pilarski et al., 1996). A B cell isdefinitively identified by its rearranged immunoglobulin genes andusually by the expression of Ig mRNA. To confirm that the CD19+ PBMCobtained by cell sorting are in fact B cells, single CD19+ PBMC from MMpatient JOD were examined for rearranged Ig genes. Single CD19+ cellswere sorted into PCR tubes and the DNA encoding the Ig heavy chain wasamplified using PCR with hemi-nested consensus framework primers to FR2,JH1 and JH2 (Table 1, SEQ ID NOS.5-7). A product of the expected size,about 160 base pairs, was amplified from patient JOD (FIG. 2), whosePBMC comprised 30% CD19+ cells. Sequencing followed by BLAST searchindicated that all bands had an IgH V region sequence. Single bonemarrow plasma cells from the same patient were also amplified using thesame consensus primers. All plasma cells examined, 11/11, gave bandsthat comigrated with those produced upon amplification of the CD19+cells (FIG. 2). Thus, all CD19+ PBMC and all BM plasma cells analyzedhad a rearranged IgH CDR3.

Identification of the Clonotypic Rearrangement.

To confirm that the product amplified in single cell PCR was in factIgH, and to determine whether or not the rearranged IgH CDR3 sequence ofBM plasma cells was shared by CD19+ PBMC, the bands shown in FIG. 2 aswell as additional bands from another gel, were excised and sequencedfrom both the FR2 and JH directions. The BM plasma cell clonotypicrearrangement for patient JOD was identified as that sequence shared bythe majority of plasma cells examined. Ten out of eleven of theindividual BM plasma cells had an identical IgH rearrangement, providingproof that this was the monoclonal JOD myeloma rearrangement (FIG. 3A).This rearrangement aligned with the DP31 VH3 gene family (FIG. 3A). Ofthe CD19+ PBMC B cells from JOD-5 (taken at month 7 after diagnosis),9/10 had an IgH CDR3 sequence identical to that of the autologous BMplasma cells with little or no intraclonal variation. RepresentativePBMC B and BM plasma cell sequences are presented in FIG. 3A (SEQ IDNO.67-70) aligned with the DP-31 sequence (SEQ ID NO.66). One B cell hadan unrelated sequence that was of a different V gene family (VH4a) (FIG.3B). The unrelated BM plasma cell aligned with the DP-31 VH3 sequence,but had low homology to the JOD consensus sequence (FIG. 3B). Thus, forpatient JOD, who had just completed 6 cycles of VAD chemotherapy, 9/10,or 90%, of blood B cells were clonotypic. The absolute number of B cellsin blood for sample JOD-5 was 0.58×10⁹/L of blood, and of these0.52×10⁹/L were clonotypic. Thus clonotypic cells in blood represented8.6% of total white blood cells.

The clonotypic PCR primers for JOD were prepared based upon the sequenceinformation obtained from the single R,and are shown in Table 1 (SEQ IDNOS.8-11).

To confirm that the sequences identified as clonotypic for JOD wereactually expressed as mRNA, sorted B cells (CD19+) were analyzed usingin situ RT-PCR to amplify the clonotypic sequence using patient specificprimers (hereinafter referred to as patient-specific amplification,PSA). Sorted B and T cells were analyzed from patient JOD-6, at 11months post diagnosis (5 months after cessation of chemotherapy). Toconfirm specificity of the JOD PSA, B and T cells from an unrelated MMpatient were also tested. All circulating B cells expressed CD19 mRNA.For two separate aliquots of JOD-6 B cells, a mean of 71% expressedclonotypic mRNA (Table 2). T cells from JOD-6 had 1% of clonotypiccells, indicating a low level of contamination by B cells. However, Tcells from two different unrelated patients had less than 0.1%clonotypic cells, and B cells from unrelated patients had 4% or lesspositive cells, perhaps reflecting limited diversity in the few normal Bcells circulating in MM (Pilarski et al., 1984; Pilarski et al., 1985),and confirming the specificity of the patient-specific primers for JOD.Thus, clonotypic B cells, as defined by patient-specific IgHrearrangement and mRNA synthesis, are not eradicated by chemotherapy,and persist for prolonged periods after cessation of therapy. For JOD-6,B cells numbered 0.22×10⁹/L of blood , and 0.15×10⁹/L were clonotypic(3.3% of total WBC). As further confirmation that patient sample JOD-6had circulating clonotypic B cells at 5 months post-chemotherapy, DNAfrom single sorted B cells was amplified by PSA using CDR2/CDR3 primers.Nine out of 27 individual B cells bad a DNA rearrangement that wasamplified by the JOD PSA to give products of the expected size, 160 basepairs, as shown in FIG. 4A.

The bands amplified in FIG. 4A were sequenced to confirm that theyindeed represented the JOD clonotypic sequence, thus validating the useof PSA in quantitating clonotypic cells in blood After amplificationwith primers to CDR3 and the 5′ terminus of CDR2 from JOD, the amplifiedproducts from 5/5 individual JOD-6 B cells had nearly identicalsequences in the intervening FR2 region, and identical sequences fromthe 25 base pairs of CDR2 not part of the primer sequence (FIG. 4B (SEQID NO.75)). Thus PSA is specific. Using PSA, a product of the expectedsize was amplified from bulk DNA isolated from BMC taken at diagnosis(JOD-4), from the staging BMC (JOD-5) and from a stable phase BMC(JOD-7), as well as from PBMC B cells but not from PBMC T cells fromJOD-7, indicated persistence of the clonotypic sequence for over a yearpost-diagnosis (data not shown).

EXAMPLE 2 PATIENT LAR

Identification of the Clonotypic Sequence for LAR.

Individual BM plasma cells from newly diagnosed MM patient LAR weresorted into PCR tubes and the expressed IgH allele was amplified inRT-PCR using the consensus framework IgH primers shown in Table 1 (SEQID NOS.5-7). A product of the expected size was amplified in 36/36individual BM plasma cells (FIG. 5A). Sequencing of the amplified bandsfrom 3 of these plasma cells indicated that they had an identical VDJsequence, shown in FIG. 5B (SEQ ID NO.76), that aligned with DP-79 ofthe VH4 family. CDR2/CDR3 LAR-specific primers, designed based on theplasma cell sequence, were used to amplify the mRNA from 48 additionalBM plasma cells; 42/48 amplified a product of the expected size, shownin FIG. 5C, confirming the identity of the clonotypic sequence in themajority (48/51 or 88%) of individual plasma cells.

Quantitation of the Proportion of Clonotypic Cells Using PCR and in situRT-PCR

To quantitate the proportion of blood B cells with clonotypic DNArearrangements, single cell PSA PCR was used to amplify DNA from LAR-1CD19+ B cells using LAR-specific primers. A clonotypic product wasamplified from 4/12 (33%) individual LAR B cells. This sample was takenat diagnosis prior to initiation of treatment. To quantitate the numberof LAR peripheral blood B cells expressing clonotypic mRNA sequences, insitu LAR PSA was used to amplify clonotypic mRNA from LAR B cells and,as a control, LAR T cells. The results are shown in Table 3. Allaliquots of B and T cells expressed histone mRNA, a housekeeping geneproduct which was used to provide a measure of the number of cells whichhad intact and detectable mRNA. For the B cell aliquots, all B cellsexpressed CD19 mRNA, as well as IgH mRNA (detected by consensusprimers). For LAR-1, in situ RT-PCR with LAR CDR2/CDR3 primers amplifieda product in 522/1183 (44%) of sorted CD19+ blood B cells. This confirmsat the mRNA level the finding that 4/12 LAR-1 B cells had clonotypic DNArearrangements. T cells from LAR, not expected to express rearrangedIgH, had less than 0.1% positive cells. For LAR-3, 46% of blood B cellswere clonotypic (Table 3). LAR-1 was taken 2 months before initiation oftherapy; LAR-3 was taken one month after initiation of therapy. Thus,for this patient, chemotherapy did not eradicate clonotypic blood Bcells. Overall, the number of clonotypic B cells was reducedapproximately 2 fold by one cycle of chemotherapy (from 0.12×10⁹/L to0.06×10⁹/L).

Quantitation of Clonotypic Cells Using Unfractionated WBC.

In order to accurately determine the total number of circulatingclonotypic B cells in MM, as well as to design a PSA assay which wouldbe easily adaptable for clinical use, in situ RT-PCR using consensus IgHprimers, as well as PSA with CDR2/CDR3 primers was performed on whiteblood cells prepared by the whole blood lysis method. The results areshown in Table 4. As predicted by previous work (Bersagel et al., 1995),approximately 3-10% of total WBC express CD19 and IgH mRNA. In situRT-PCR amplification with IgH VDJ consensus primers of mRNA in WBC gavea mean of 5% for 5 different patients, with a range of 3.3% to 7.4%. Useof CD19 primers to amplify mRNA from WBC gave a mean value of 6.8% for 5different patients, with a range of 2.5% to 10.4% of WBC. To determinethe frequency of clonotypic cells among unfractionated WBC, PSA in situRT-PCR with LAR primers was used. For LAR-3, in situ RT-PCR usingunfractionated WBC prepared by the red cell lysis method for WBCdemonstrated that 145/2116 WBC, or 6.8% of total WBC were CD19+. Todetermine the number of circulating clonotypic cells, LAR WBC wereamplified using PSA with CDR2 and CDR3 primers. By this measure, 24/1987WBC, or 1.2% of LAR-3 WBC were clonotypic, confirming calculations fromTable 3. The infrequent presence of either LAR or JOD sequences amongWBC from unrelated MM patients confirms the specificity of the assay.The simplicity and quantitative nature of PSA using in situ RT-PCR ontotal WBC makes this approach feasible in a clinical laboratory, formonitoring circulating relatives of the malignant myeloma clone duringtreatment. It is also a validation of more complex methods fordetermining the frequency of clonotypic B cells that rely on purified Bcell populations. Calculated values from purified populations appear toreasonably estimate the absolute numbers obtained by analysis ofunmanipulated WBC.

Specificity of JOD and LAR PSA.

Total RNA from sorted LAR B cells, T cells and BM plasma cells, as wellas BM plasma cells from 2 unrelated MM patients, was amplified inconventional bulk RT-PCR. An amplified product of the expected size wasdetected in B and plasma cells from LAR, but was absent from LAR T cellsand from BM plasma cells of two unrelated MM patients (FIG. 6),confirming the specificity of the LAR primers, and the presence ofclonotypic mRNA within the B cell population. The identity of the PSART-PCR product for LAR B and plasma cells was confirmed by analysis ofthe products using restriction fragment length polymorphism (not shown).Control experiments also demonstrated that JOD primers did not amplify aproduct from LAR blood subsets of BMC in bulk RT-PCR, and LAR primersdid not amplify a band from JOD-5, JOD-6 or JOD-7 PBMC, PBMC subsets orBMC (not shown). Results identical to those for LAR-1 showing thepresence of clonotypic sequences in BMC and PBMC B cells, but not PBMC Tcells, were obtained for LAR-3 using bulk PSA of mRNA (not shown).

Discussion of Results.

The above examples demonstrate that in MM patients, 44-90% of totalcirculating blood B cells, or 1-9% of total WBC have an IgHrearrangement identical to that of autologous BM plasma cells, the tumorcells. The absolute number ranges from 0.06-0.5×10⁹ clonotypic cells/Lof blood. An important aspect of this work is the identification of theclonotypic IgH sequence as that expressed by the majority of individualBM plasma cells in a patient. This type of quantitation is possible onlyat the single cell level, either by single cell PCR or RT-PCR, or by insitu RT-PCR. Although previous studies analyzed homogenized preparationsof nucleic acid derived from unfractionated bone marrow, the assumptionwas made that most plasma cells express the IgH sequence identified aspredominant in bulk PCR or RT-PCR. Confirmation of this was essential,since only an IgH CDR3 sequence that identifies most BM plasma cellswould be expected in the blood. Using single cell PCR or RT-PCR, forpatient JOD and LAR, 10/11 and 45/51 respectively of BM plasma cellsexpressed the sequence identified as clonotypic. A second refinement inthis work was the use of two patient specific primers, CDR2 and CDR3,for amplification of clonotypic sequences. The inventors found that useof a primer specific for patient-specific CDR3 together with a consensusFR2 primer, as commonly used in other studies (allele-specific oligomer(ASO)-PCR), was less specific than was the use of PSA with CDR2/CDR3primers to drive high stringency amplification of IgH variable regions.The patient-specific amplification (PSA) methods were validated by theinventors demonstration that the sequences of CDR2/CDR3 amplifiedproducts from 6 individual B cells were clonotypic. For patient JOD, thepresence of the clonotypic sequence was confirmed in 3 different samplesof BM plasma cells taken over a 1.3 year period including at diagnosis(JOD-4), for staging after chemotherapy (JOD-5), and during the stablephase of disease (JOD-7). The clonotypic JOD sequence was detectable inPBMC or in purified B cells from 4 sequential JOD samples.

For all patients tested, all CD19+ (by cell sorting) blood B cellsexpressed both CD19 mRNA and IgH mRNA. Based on mean values fromphenotypic analysis of nearly 500 patients done previously, the absolutenumbers of circulating blood B cells represent about 3-10% of total WBC,or about 0.4×10⁹ B cells/L of blood (unpublished results and Bergsagelet al., 1995). For patient JOD at 1 month post-chemotherapy (JOD-5), 90%of individual circulating B cells hare an IgH VDJ rearrangementidentical to that of autologous BM plasma cells. However, for JOD-6,taken 5 months after cessation of therapy, the absolute number andproportion of clonotypic B cells decreased, suggesting recovery ofpolyclonal B cells post-chemotherapy, and by extrapolation thatchemotherapy may actually enrich for clonotypic B cells, althoughabsolute numbers may decrease. This may reflect a greaterchemosensitivity for normal B cells as compared to the clonotypic set.For patient LAR-1, at diagnosis, 44% of circulating B cells wereclonotypic. After one cycle of chemotherapy, the proportion ofclonotypic B cells remained constant, although absolute numbersdecreased suggesting depletion after the initial exposure tochemotherapy.

For patient LAR, newly diagnosed, about 44% of total CD19+ B cells areclonotypic. For JOD, after 6 cycles of chemotherapy, nearly all B cells(90%) were clonotypic. At 5 months (JOD-6) post-chemotherapy, 71% ofblood B cells were clonotypic, indicating their persistence afterchemotherapy. Calculations of the absolute number of clonotypic B cellscirculating the blood of these two patients give values of 1-9% of whiteblood cells or 0.06-0.52×10⁹ clonotypic B cells/L of blood comparisonwith normal values indicates expanded numbers of overall B cells, andenriched proportions of monotypic B cells. Sequential samples for bothJOD and LAR indicate that chemotherapy does not effectively targetcirculating clonotypic B cells.

The long term goal of this work is to evaluate the link betweenclonotypic B cells, disease progression and spread, and clinicaloutcome. The frequent relapse rate for myeloma indicates that currentmodes of treatment are not accompanied by elimination of the generativecompartment of MM. PSA for monitoring clonotypic cells in whole bloodusing in situ RT-PCR or limiting dilution PCR with patient-specificprimers proves a clinically feasible monitoring strategy for determiningthe extent to which clonal cells infiltrate the blood of MM patientsbefore, during and after therapy, as well as their persistence aftercytoreduction and transplantation. Measurement of circulating clonotypicB cell numbers using in situ PSA provides a marker of blood involvementthat complements measure of plasma cell kill, to evaluate the efficacyof present and future therapies which target the malignant clone in MM.This assay allows the use of blood tests rather than the more invasiveand expensive bone marrow tests for routine clinical monitoring. Whenbone marrow or tissue samples are necessary, this assay is quantitative,more specific and more sensitive than any currently available test.

EXAMPLE 3 ANALYSIS OF ADDITIONAL MYELOMA PATIENTS

The aforementioned methods for determining the frequency of clonotypic Bcells in blood was applied to a group of 18 multiple myeloma patients.This example clearly demonstrates how the methods of the invention areused to monitor the number of clonotypic B cells before, during andafter treatment by chemotherapy or hematopoietic transplantation.

The characteristics of the clonotypic sequences derived from BM plasmacells of the patients are presented in Tables 4 and 5. The patients havethe Ig isotype distribution characteristic of myeloma. The pattern of Vhfamily usage included frequent use of the Vh3 gene family and infrequentuse of the Vh1 family (Table 4). The most frequent J segment was Jh4(65% of the patients). Overall, the CDR3 length was 12-54 nucleotides(NT) (mean +/−SE=28+/−2 NT). The sequences of the CDR3 portion of IgHare presented in Table 5 (SEQ ID NOS.30-47).

9-90% of Myeloma PBMC B Cells are Clonotypic as Detected by in situRT-PCR

For 17 myeloma patients (one patient from Table 4 died prior to thisanalysis) and 35 blood samples analyzed in this study, the percentage ofcirculating B cells expressing clonotypic mRNA was on average 66%. Table6 records the frequency of clonotypic B cells for each myeloma patientfor one or more blood samples taken at regular clinical visits. In allcases the sequences identified as clonotypic were confirmed to beexpressed by >80% of autologous BM plasma cells. In blood, theproportion of B cells expressing clonotypic IgH mRNA ranged from 9-90%with a mean of 66%+/−4% (SE). These values were used to calculate that14+/−2% of PBMC were clonotypic cells (range=0.9-50% of PBMC)

The proportion of clonotypic cells among total white blood cells wascalculated as 3.5+/−1% (range=1-9%) (data not shown). The absolutenumber of circulating clonotypic B cells was 0.15+/−0.02×10⁹/L of blood(range=0.01-0.61×10⁹/L).

Abbreviations BMC bone marrow cells dNTP deoxynucleotide triphosphatesPBMC peripheral blood mononuclear cells MGUS monoclonal gammopathy ofundeter- mined significance MM multiple myeloma IgH immunoglobulin heavychain VDJ variable, diversity, joining WBC white blood cells PSApatient-specific amplification TCR T cell receptor FITC Fluoresceneisothiocyanate PE phycoerytherin MW molecular weight BM bone marrow ALLAcute Lymphocytic Leukemia JOD, LAR, JES, BTA Nomenclature forindividual patients CLL Chronic Lymphocytic Leukemia VAD Vincristine,Adriamycin, Dexametha- sone M Melphalan Dex Dexamethasone UV Ultraviolet

TABLE 1 Primers used for in situ RT-PCR, for PCR and for RT-PCR Histone5′ CCACTGAACTTCTGATTCGC Histone 3′ GCGTGCTAGCTGGATGTCTT CD19 5′GACCTCACCATGGCCCCTGG CD19 3′ CAGCCAGTGCCATAGTAC Consensus IgH primersIgH FR2 5′ TATGAATTCGGAAAGGGCCTGGAGTGG IgH JHI 3′ACGGGATCCACCTGAGGAGACGGTGACC IgH JH2 3′ ACGGATCCGTGACCAGGGTNCCTTGGCCCCAGPatient-specific CDR2/CDR3 primers for PSA: JOD5 CDR2 5′CGTGGAATAGGGGCAGTC JOD5 CDR3 3′ AAGTTGTAGCCATCTCGG LAR1 CDR2 5′ACTTCTACGACAATGGCGAAAC LAR1 CDR3 3′ CCCTCCGAGGACGTGGTG Note: the abovesequences represent SEQ ID NOS: 1-11, respectively.

TABLE 2 PBMC B cells, but not T cells, express clonotypic sequences forpatient JOD, after chemotherapy Clonotypic B Cells % Clonotypic # ×10⁹/L % of WBC Patient Tissue Subset (# counted) of Blood CalculatedJOD-5 BMC CD38⁺Ig⁺ 91 (10/11) JOD-5 PBMC CD19⁺ 90 (9/10) 0.52 9 JOD-6PBMC CD19⁺ 71 (1111/1555) 0.15 3 1.3 (6/463) NKI-7 PBMC CD19⁺ 3.7(12/305) 0.007 0.06 CD3⁺ <0.1 (0/1000) RAM-9 PBMC CD19⁺ <0.1 (0/1000)<0.0006 <0.01 CD3⁺ <0.1 (0/1000)

PBMC or BMC were sorted as indicated and analyzed using PSA with in situRT-PCR with primers specific for CDR2 and CDR3 of JOD (lines 3-11), orsequencing (lines 1 and 2). For sorted CD19+ PBMC from unrelatedpatients, the presence of B cells was confirmed using in situ RT-PCR forCD 19, and viability of mRNA was established using histone primers.

TABLE 3 PBMC B cells, but not T cells, have clonotypic IgH VDJ sequencesfor patient LAR, at diagnosis and after initiation of treatmentClonotypic B Cells Subset # × 10⁹/L % of WBC Patient Tissue (Sorted) %Clonotypic of Blood (Calculated) LAR-1 BMC CD38⁺Ig⁺ 88 (45/51) LAR-1PBMC CD19⁺ 44 (522/1183) 0.12 × 10⁹ 2.2 CD3⁺  1 (5/452) LAR-3 PBMC CD19⁺46 (190/409) 0.06 × 10⁹ 1.1 CD3⁺ <0.1 (0/1000)

PBMC and BMC were analyzed using PSA with CDR2 and CDR3 primers frompatient LAR in in situ RT-PCR. In all cases, viability of mRNA wasconfirmed by amplification with primers to histone.

TABLE 4 Characteristics of Clonotypic IgH VDJ sequences Patient % Vh JhCDR3 (status*) IgH Lt PC^(a) Family Family Length (NT)  1. (Unt) IgG K23 Vh2(S12-12) Jh4(b) 27  2. (Unt) IgG K 31 Vh5(DP73) Jh3(a) 18  3. (Tr)IgA K 33 Vh3(DP77) Jh2  6  4. (Unt) IgA K 30 Vh4(DP65) nd 21  5. (Unt)IgG L 23 Vh3(DP49) Jh6(b) 30  6. (Unt) IgG K 30 Vh4(DP71) Jh4(a) 36  7.(Unt) IgG K 25 VH3(DP77) Jh2 30  8. (Off) IgA L 57 Vh3(DP 46) Jh3(b) 21 9. (Off) Nd^(a) L 80 Vh3-21(DP77) Jh6(c) 24 10. (Unt) IgG K 90Vh2(S12-12) Jh4(a) 33 11. (Sm) IgA K 51 Vh3-30(DP49) Jh4(a) 18 12.(Tr)** IgG L 11 Vh3-15(DP38) Jh4(a) 21 13. (Unt) IgA K 70 Vh3-8(DP58)Jh4(a) 27 14. (Off)*** IgG K 67 Vh3-30(DP49) Jh4(b) 24 15. (Unt) IgG K75 Vh4-31(DP78) Jh4(b) 12 16. (Unt) IgG K 22 Vh1(DP88) Jh4(b) 36 17.(Unt) IgG K 50 Vh3-49(DP57) Jh4(c) 54 18. (Tr) IgG K 41 Vh5-51(DP73)Jh4(b) 15 *These patients were all diagnosed with myeloma; patients #8and 14 were in relapse at the time their BM was obtained. Patient #10died one month post-diagnosis. Their treatment status at the time thebone marrow sample was obtained is indicated. Patient 11 has smolderingmyeloma, and remains untreated, for the samples analyzed here. Selectioncriteria for this study were that the patient was diagnosed with myelomaand that # a fresh BM sample was available; no selection was applied forstage of disease or treatment status. **This patient was in stable phaseimmediately prior to hematopoietic transplantation. ***This patient wasfirst diagnosed in 1988 and is thus a long term survivor. In 1993/94,she had clonotypic DNA sequences detectable in her blood (6, Patient #3in that study). The sequence of IgH VDJ transcripts in her plasma cellswas determined at relapse for this study (in 1997) using single sortedBM plasma cells. The CDR3 sequence obtained was identical to thatdetermined previously (6). All of her relapse plasma cells express thissequence. ^(a)% PC = percent of plasma cells in the bone marrow sampleused to derive the clonotypic sequence; ND = not detectable by routineclinical methods; Lt = light chain; K = kappa; L = lambda; Unt =untreated; Tr = treated; and Off = off therapy.

TABLE 5 Clonotypic IgH CDR3 sequences and primers used for PSA (SEQ IDNOS: 12-29) (SEQ ID NOS: 30-47) codon 92 FR3 CDR3 01 TGTGCTCACAAACTTATCACTGGTTGGGACGGTAGTAGT 02 TGTGCGACA CAACACTACTATGATAGT 03TGTGCGAGA GATACCTATTATTATGGTTCAGGGAGTTATTCA 04 TGTGCGGGTGGCACCACGTCCTCCCAGGGT 05 TGTGCGAAG CTCGTGGTTGTGGCGGTGGAAGCTCTAACCCAT 06TGTGCGAGG GTCCCCATGAACTATGCTATAAGGGGAAACTTAGGT 07 TGTGCGAGAGAATGGTCGTACTTCTATGAAAGTTATTGGTTA 08 TGTGCGAGAGACGGAAGCAGAGATGGCTACAACTCG 09 TGTGCGAGA GGGGATGGTTCGGGAGAGATCTTT 10TGTTCACAC ACGCGTTTCATGCCTGCGGATGTGAACAACTTC 11 TGTGCGCCAGTTCTTGCCAACTGGTTT 12 TGTACCACA GCGTTCAGTGAGCCCTCCAGC 13 TGTGCGACAGATCAAGATGACTATGGTGACTACGGGACC 14 TGTACGAGAGTAAATCCTTTCTATGAAGGTAGTCGTTATCCCATA 15 TGCGCCACA GATCCCTCTGAC 16TGTGCGACA GTAAATCCTTTCTATGAAGGTAGTCGTTATCCCATA 17 TGTACTAGAGATAGGGAGGATACTGTAGTAGGAACAGTTACTATGGGCCGAATACCCACGGTT 18 TGTGCGAGACATTATCACGGTTAC (SEQ ID NOS: 48-65) codon 92 JH 01 TACTTTGACCAG.TGGGGC02 TATATTGACTTC.TGGGGC 03 TACTGGTACTTCGATCTC.TGGGGC 04CAGAGGTTGGAACTC.TGGGGC 05 GATAATCTTGATATT.TGGGGC 06 TCCATTGACTAC.TGGGGC07 TTACCCTTTGACTTC.TGGGGC 08 GGTGTTTTTGATATC.TGGGGC 09CCTTACTACTACTATCACATGGACGTC.TGGGGC 10 TTTGACTAC.TGGGGC 11CGCCCCTTFGACCAC.TGGGGC 12 GACTACTACACGATGGACTTC.TGGGGC 13TTFAACTCC.TGGGGC 14 TACTACTTTGGCFAC.TGGGGC 15 TACTTFGACCTC.TGGGGC 16TACTACTTFGGCTAC.TGGGGC 17 AAATACTACTACTACCACCACATGGACGTC.TGGGGC 18CGATCGGACGTC.TGGGGC

Regions used as patient-specific CDR3 primers are highlighted in bolditalics.

TABLE 6 Clonotypic B cells are frequent in the blood of myeloma patients# of Patient sequential % of cells that are clonotypic Status- timeAbsolute # × 10⁹/L Apr. 97 points of B cells of PBMC Blood  1. Tr 569,99,91 24,3,14 0.1,0.35,0.09 95,62 10,18 0.05,0.11  2. Deceased 274,74 19,31 0.17,0.15  3. Tr 2 71,54 14,9 0.22,0.12  4. Allo Tsp 445,46,46,62 9,9,10,19 0.12,0.08,0.06,0.11  5. Off 3 91,40,73 18,8,160.24,0.1,0.21  6. Tr 3 60,77,90 10,10,50 0.08,0.03,0.1  7. Deceased 1 9312 0.06  8. Deceased 1 9 0.9 0.01  9. Tr 1 52 7 0.09 11. Unt 2 65,6410,6 0.08,0.06 12. Auto Tsp 2 46,64 15,7 0.16,0.13 13. Tr 1 73 12 0.1814. Tr 2 28,90 8,21 0.03,0.08 15. Tr 2 66,90 11,13 0.17,0.13 16. Tr 1 5613 0.17 17. Tr 1 53 20 0.42 18. Tr 2 68,49 20,18 0.61,0.39 Mean 66 ± 414 ± 2 0.15 ± .02

Normal PBMC (15 donors)

CD19⁺ PBMC (60 slides) <0.3%

Normal Plasma Cells (BM, 4 Donors)

CD38^(hi)Ig⁺BMC (19 slides) <0.3%

Individual time points are listed sequentially in the table. #1 wasslowly responding to melphalan (M) and dexamethasone (Dex), #2 hadM/Dex, #3 is relapsing after treatment with M/prednisone (P), #4 wastreated with M/P and then received an allogeneic transplant, #5 is beingtreated with M/P, #6 was treated with M/Decadron but did not respond, #7received M/Dex, #8 was at a terminal stage of disease, #9 was newlydiagnosed untreated, #11 has smoldering myeloma and remains untreated,#12 responded to VAD and was in plateau, and #13 was newly diagnoseduntreated. Patient #14 was in relapse. Patients #15-18 were studied atdiagnosis and/or during the first 1-2 cycles of first line therapy. Meanvalues are ±SE. For all samples, a minimum of 300 cells and frequently500-1000 cells were viewed. In all cases, for in situ RT-PCR assaysbeing performed on a given day, the patient-specific primers beingtested each day on sorted myeloma B cells were also tested on B cellsfrom normal donors to confirm their specificity. Every set ofpatient-specific primers has been tested on sorted B cells from at least2 normal donors and on sorted CD19+ BMC from at least one normal donor.No amplification was detected in these normal donor controls.

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76 1 20 DNA Homo sapiens 1 ccactgaact tctgattcgc 20 2 20 DNA Homosapiens 2 gcgtgctagc tggatgtctt 20 3 20 DNA Homo sapiens 3 gacctcaccatggcccctgg 20 4 18 DNA Homo sapiens 4 cagccagtgc catagtac 18 5 27 DNAHomo sapiens 5 tatgaattcg gaaagggcct ggagtgg 27 6 28 DNA Homo sapiens 6acgggatcca cctgaggaga cggtgacc 28 7 32 DNA Homo sapiens misc_feature 20n = a, c, g, or t 7 acggatccgt gaccagggtn ccttggcccc ag 32 8 18 DNA Homosapiens 8 cgtggaatag gggcagtc 18 9 18 DNA Homo sapiens 9 aagttgtagccatctcgg 18 10 22 DNA Homo sapiens 10 acttctacga caatggcgaa ac 22 11 18DNA Homo sapiens 11 ccctccgagg acgtggtg 18 12 9 DNA Homo sapiens 12tgtgctcac 9 13 9 DNA Homo sapiens 13 tgtgcgaca 9 14 9 DNA Homo sapiens14 tgtgcgaga 9 15 9 DNA Homo sapiens 15 tgtgcgggt 9 16 9 DNA Homosapiens 16 tgtgcgaag 9 17 9 DNA Homo sapiens 17 tgtgcgagg 9 18 9 DNAHomo sapiens 18 tgtgcgaga 9 19 9 DNA Homo sapiens 19 tgtgcgaga 9 20 9DNA Homo sapiens 20 tgtgcgaga 9 21 9 DNA Homo sapiens 21 tgttcacac 9 229 DNA Homo sapiens 22 tgtgcgcca 9 23 9 DNA Homo sapiens 23 tgtaccaca 924 9 DNA Homo sapiens 24 tgtgcgaca 9 25 9 DNA Homo sapiens 25 tgtacgaga9 26 9 DNA Homo sapiens 26 tgcgccaca 9 27 9 DNA Homo sapiens 27tgtgcgaca 9 28 9 DNA Homo sapiens 28 tgtactaga 9 29 9 DNA Homo sapiens29 tgtgcgaga 9 30 30 DNA Homo sapiens 30 aaacttatca ctggttgggacggtagtagt 30 31 18 DNA Homo sapiens 31 caacactact atgatagt 18 32 33 DNAHomo sapiens 32 gatacctatt attatggttc agggagttat tca 33 33 21 DNA Homosapiens 33 ggcaccacgt cctcccaggg t 21 34 33 DNA Homo sapiens 34ctcgtggttg tggcggtgga agctctaacc cat 33 35 36 DNA Homo sapiens 35gtccccatga actatgctat aaggggaaac ttaggt 36 36 33 DNA Homo sapiens 36gaatggtcgt acttctatga aagttattgg tta 33 37 27 DNA Homo sapiens 37gacggaagca gagatggcta caactcg 27 38 24 DNA Homo sapiens 38 ggggatggttcgggagagat cttt 24 39 33 DNA Homo sapiens 39 acgcgtttca tgcctgcggatgtgaacaac ttc 33 40 18 DNA Homo sapiens 40 gttcttgcca actggttt 18 41 21DNA Homo sapiens 41 gcgttcagtg agccctccag c 21 42 30 DNA Homo sapiens 42gatcaagatg actacggtga ctacgggacc 30 43 36 DNA Homo sapiens 43 gtaaatcctttctatgaagg tagtcgttat cccata 36 44 12 DNA Homo sapiens 44 gatccctctg ac12 45 36 DNA Homo sapiens 45 gtaaatcctt tctatgaagg tagtcgttat cccata 3646 54 DNA Homo sapiens 46 gatagggagg atactgtagt aggaacagtt actatgggccgaatacccac ggtt 54 47 15 DNA Homo sapiens 47 cattatcacg gttac 15 48 18DNA Homo sapiens 48 tactttgacc agtggggc 18 49 18 DNA Homo sapiens 49tatattgact tctggggc 18 50 24 DNA Homo sapiens 50 tactggtact tcgatctctggggc 24 51 21 DNA Homo sapiens 51 cagaggttgg aactctgggg c 21 52 21 DNAHomo sapiens 52 gataatcttg atatttgggg c 21 53 18 DNA Homo sapiens 53tccattgact actggggc 18 54 21 DNA Homo sapiens 54 ttaccctttg acttctgggg c21 55 21 DNA Homo sapiens 55 ggtgtttttg atatctgggg c 21 56 33 DNA Homosapiens 56 ccttactact actatcacat ggacgtctgg ggc 33 57 15 DNA Homosapiens 57 tttgactact ggggc 15 58 21 DNA Homo sapiens 58 cgcccctttgaccactgggg c 21 59 27 DNA Homo sapiens 59 gactactaca cgatggactt ctggggc27 60 15 DNA Homo sapiens 60 tttaactcct ggggc 15 61 21 DNA Homo sapiens61 tactactttg gctactgggg c 21 62 18 DNA Homo sapiens 62 tactttgacctctggggc 18 63 21 DNA Homo sapiens 63 tactactttg gctactgggg c 21 64 36DNA Homo sapiens 64 aaatactact actaccacca catggacgtc tggggc 36 65 18 DNAHomo sapiens 65 cgatcggacg tctggggc 18 66 153 DNA Artificial SequenceDescription of Artificial Sequence primer 66 gtctcaggta ttagttggaatagtggtagc ataggctatg cggactctgt gaagggccga 60 ttcaccatct caagagacaacgccaagaac tccctgtatc tgcaaatgaa cagtctgaga 120 gctgaggaca cggccttgtattactgtgca aaa 153 67 153 DNA Artificial Sequence Description ofArtificial Sequence primer 67 gtctcaggta ttacgtggaa taggggcagtctaggatatg tggactctgt caggggccga 60 ttcaccatct ccaaagacag cgtgaagaagttcctgtatc tgcaaatgaa cagtctgaga 120 actgaggaca cggccttgta ttattgtgtaaag 153 68 153 DNA Artificial Sequence Description of ArtificialSequence primer 68 gtctcaggta ttacgtggaa taggggcagt ctaggatatgtggactctgt caggggccga 60 ttcaccatct ccaaagacag cgtgaacaag ttcctgtatctgcaaatgaa cagtctgaga 120 actgaggaca cggccttgta ttattgtgta aag 153 69153 DNA Artificial Sequence Description of Artificial Sequence primer 69gtctcaggta ttacgtggaa taggggcagt ctaggatatg tggactctgt caggggccga 60ttcaccatct ccaaagacag cgtgaagaag ttcctgtatc tgcaaatgaa cagtctgaga 120actgaggaca cggccttgta ttattgtgta aag 153 70 153 DNA Artificial SequenceDescription of Artificial Sequence primer 70 gtctcaggta ttacgtggaataggggcagt ctaggatatg tggactctgt caggggccga 60 ttcaccatct ccaaagacagctgtaagaag ttcctgtatc tgcaaatgaa cagtctgaga 120 actgaggaca cggccttgtattatttgtta aag 153 71 184 DNA Artificial Sequence Description ofArtificial Sequence consensus sequence 71 gtctcaagta ttacgtggaataggggcagt ctaggatatg tggactctgt caggggccga 60 ttcaccatct ccaaagacagcgtgaagaag ttcctgtatc tgcaaatgaa cagtctgaga 120 actgaggaca cggccttgtattattgtgta aaggccgaga tggctacaac ttcgaaggac 180 aaca 184 72 179 DNAArtificial Sequence Description of Artificial Sequence consensussequence 72 gtctcaagta ttacgtggaa taggggcagt ctaggatatg tggactctgtcaggggccga 60 ttcaccatct ccaagacagc gtgaagaagt tcctgtatct gcaaatgaacagtctgagaa 120 ctgaggacac ggccttgtat tattgtgtaa aggccgagat ggctacaacttcgaaggac 179 73 161 DNA Artificial Sequence Description of ArtificialSequence primer 73 gtcaccgtct cctcaggtgg atcccgtacc tggggccaagggactggtca cggatcccgt 60 aaccttatga attcggaaag ggcctggagt gggtctcaagtattacgtgg aataggggca 120 gtctaggata tgtggactct gtcaggggcc gattcaccat c161 74 187 DNA Artificial Sequence Description of Artificial Sequenceprimer 74 tcaccgtcct acgaagtcgg ataccgtctg gagtggccac cgtctcgtcaggtatatccc 60 gaactggggc aatggcacct ggtcacggat cggttatgaa ttcggaatgggcctggagtt 120 gtctcgtatt cctgaacgtg gttcccgtcc ccttatgaat tcggaaagggcctggagtgg 180 tcaccgt 187 75 121 DNA Artificial Sequence Description ofArtificial Sequence consensus sequence 75 tggaataggg gcagtctaggatatgtggac tctgtcaggg gccgattcac catctccaaa 60 gacagcgtga agaagttcctgtatctgcaa atgaacagtc tgagaactca ggacacggcc 120 t 121 76 186 DNAArtificial Sequence Description of Artificial Sequence primer 76cttgggactt ctacgacaat ggcgaaacct tcaacaatcc gtcctcacgg gtggtcgagt 60caccgtgtcc ctagacacat ctcagaatta tttgtccctg gaagtagtct ctgtgaacgc 120cgcagacacg ggtatttatt actgtgcggg tggcaccacg tcctcccagg gtcagaggtt 180ggaatc 186

We claim:
 1. A method for detecting a target clonotypic nucleic acidrearrangement in hematopoietic cells from a subject having, or at riskof having, a hematopoietic neoplastic disorder comprising: a) isolatinga hematopoietic neoplastic cell containing the target clonotypic nucleicacid rearrangement; b) amplifying a specific segment of the targetnucleic acid containing the clonotypic rearrangement from said isolatedhematopoietic neoplastic cell; c) determining the sequence of theamplified segment, thereby identifying a specific clonotypic nucleicacid rearrangement; and d) quantitatively detecting the presence of saidspecific clonotypic nucleic acid rearrangement in a second population ofisolated intact hematopoietic cells derived from a subject having, or atrisk of having, a neoplastic hematopoietic disorder, such that saidtarget clonotypic nucleic acid rearrangement in said second populationof isolated hematopoietic cells is detected.
 2. The method of claim 1wherein the hematopoietic cells are malignant cells.
 3. The method ofclaim 2 wherein the hematopoietic cells are B cells or T cells.
 4. Themethod of claim 1 wherein the hematopoietic cells are multiple myelomacells.
 5. The method of claim 1 wherein the clonotypic rearrangement isin an Ig gene locus.
 6. The method of claim 1 wherein the clonotypicrearrangement is in a TCR gene locus.
 7. The method of claim 1 whereinthe clonotypic rearrangement is a chromosomal translocation.
 8. Themethod of claim 1, wherein the amplifying is by PCR.
 9. The method ofclaim 8 wherein cells bearing a clonotypic rearrangement are detected bydirect labeling of a PCR product.
 10. The method of claim 1 whereincells bearing a clonotypic rearrangement are detected by nucleic acidhybridization to a PCR product.
 11. The method of claim 8 wherein PCRprimers for PCR specifically amplify the unique hypervariable regions ofthe IgH, k or 1 Ig gene, or of the TCR a, b, g or d chain.
 12. Themethod of claim 8 wherein PCR primers for PCR are specific for the CDR1,CDR2 and/or the CDR3 region.
 13. The method of claim 12 wherein thespecific primers for the CDR1, CDR2 or CDR3 region are used inconjunction with a framework-specific consensus primer.
 14. The methodof claim 1, wherein said subject is being treated for a hematologicalmalignancy, and wherein said hematopoietic neoplastic cell is isolatedfrom said subject's blood or bone marrow.
 15. The method of claim 1,wherein the specific clonotypic nucleic acid rearrangement is detectedin cells destined for autologous transplantation.
 16. The method ofclaim 1, wherein the presence of the specific clonotypic nucleic acidrearrangement is detected in intact hematopoietic cells.
 17. The methodof claim 1, wherein the presence of said specific clonotypic nucleicacid rearrangement is detected by determining the frequency of saidrearrangement in said second population of isolated intact hematopoieticcells.
 18. The method of claim 1, wherein said second population ofintact hematopoietic cells consists of a single intact hematopoieticcell.
 19. A method for determining the frequency of a target clonotypicnucleic acid rearrangement in hematopoietic cells from a subject having,or at risk of having, a hematopoietic neoplastic disorder consisting of:a) isolating a hematopoietic neoplastic cell containing the targetclonotypic nucleic acid rearrangement; b) amplifying a specific segmentof the target nucleic acid containing the clonotypic rearrangement fromsaid isolated hematopoietic neoplastic cell; c) determining the sequenceof the amplified segment, thereby identifying a specific clonotypicnucleic acid rearrangement; d) detecting the presence of said specificclonotypic nucleic acid rearrangement in an isolated intacthematopoietic cell derived from said subject; and e) detecting thepresence of said specific clonotypic nucleic acid rearrangement inadditional isolated intact hematopoietic cells, such that the frequencyof said target clonotypic nucleic acid rearrangement in said secondpopulation of isolated hematopoietic cells is determined.